identifying cancer patients from GC-patterned fragment ends of cell-free DNA

One of the most intriguing characteristics of cell-free DNA (cfDNA) from plasma is the sequence at the ends of the fragments. Previous studies have shown that these end-sequences are somewhat different in cancer patients than in healthy individuals. While investigating this characteristic, we noticed that the bases at the 5’-ends of a double-stranded fragment were highly correlated with the GC content of that particular fragment. This led us to develop a method, called MendSeqS (Modified End-based sequencing System), that incorporates the correlation between end-motifs and GC content into the analysis of shallow (0.5x) whole genome sequencing (WGS). When applied to plasma samples, MendSeqS was able to classify patients with a sensitivity of 96% at 98% specificity in a cohort comprised of 107 individuals evaluated in our laboratory (43 with cancer and 64 without). In cohorts evaluated in three other laboratories, comprising a total of 401 individuals (193 with cancer and 208 without), MendSeqS achieved a sensitivity of 87% at 98% specificity. MendSeqS could in principle be combined with other methods of cfDNA analysis to enhance cancer detection. ### Competing Interest Statement BV, KWK, & NP are founders of Thrive Earlier Detection, an Exact Sciences Company. KWK, NP, & CD are consultants to Thrive Earlier Detection. BV, KWK, NP, SZ, and CD hold equity in Exact Sciences. BV, KWK, NP, and SZ, are founders of or consultants to and own equity in ManaT Bio., Haystack Oncology, Neophore, CAGE Pharma and Personal Genome Diagnostics. NP is consultant to Vidium. PG and JT are consultants to Haystack Oncology. BV is a consultant to and holds equity in Catalio Capital Management SZ has a research agreement with BioMed Valley Discoveries, Inc. CB is a consultant to Depuy-Synthes, Bionaut Labs, Haystack Oncology and Galectin Therapeutics. CB is a co-founder of OrisDx. The companies named above, as well as other companies, have licensed previously described technologies related to the work described in this paper from Johns Hopkins University. BV, KWK, NP, JDC, and CD are inventors on some of these technologies. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors as well as to Johns Hopkins University. Patent applications on the work described in this paper may be filed by Johns Hopkins University. The terms of all these arrangements are being managed by Johns Hopkins University in accordance with its conflict of interest policies. ### Funding Statement This work was supported by The Lustgarten Foundation for Pancreatic Cancer Research, The Virginia and D.K. Ludwig Fund for Cancer Research, The Conrad N. Hilton Foundation, The Sol Goldman Charitable Foundation, and National Institutes of Health grants (T32 GM008752, U01 CA200469, U01 CA62924, T32 GM136577, U01 CA06973, T32 GM135083, and T32 GM007814). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Approval was granted by the institutional Review Board of the Johns Hopkins Medical Institutions. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The sequencing data generated in this study can be obtained from the European Genome phenome Archive (accession number EGAS00001006418)